Showing papers in "Journal of Andrology in 2000"

DNA Integrity in Human Spermatozoa: Relationships With Semen Quality

Abstract: The literature contains conflicting evidence regarding the existence of DNA damage in spermatozoa from infertile male patients. To examine this phenomenon, we have studied ejaculated spermatozoa from normozoospermic semen donors and from a group of the unselected male partners of couples attending an infertility clinic for initial investigation. Classical semen analysis according to World Health Organization (WHO) guidelines was undertaken with computer-assisted sperm analysis (CASA). Spermatozoa were prepared by sequential washing and centrifugation and were analyzed for DNA fragmentation using three assays: 1) a single-cell gel electrophoresis (comet) assay, 2) in situ nick translation with prior chemical decondensation (ISNT-decondensed), and 3) in situ nick translation without prior chemical decondensation (ISNT-condensed). In addition, reactive oxygen species (ROS) generation by spermatozoa was measured, and seminal plasma was analyzed for its total reactive antioxidant potential (TRAP). When the donor and patient groups were compared, the latter had lower levels of semen quality and higher levels of DNA damage, which was particularly apparent using the comet assay. Highly significant negative correlations were observed between DNA fragmentation, detected by all three assays, and semen quality, particularly sperm concentration. In addition, multiple regression analysis indicated that other attributes of semen quality, such as sperm movement and ROS generation, were also related to DNA damage. We conclude that a significant proportion of infertile men have elevated levels of DNA damage in their ejaculated spermatozoa.

Semen cryopreservation in domestic animals: a damaging and capacitating phenomenon.

Abstract: The genetic improvement of agriculturally important species and disease control are of fundamental importance to the success of a sustainable agri-food industry. In this sense, artificial insemination (AI) is arguably the most important tool contributing to the advancement of modern animal production. Through AI, 1 ejaculate from a genetically superior male can be used to impregnate multiple females to maximize the distribution of agriculturally favorable genes. As well, AI eliminates physical contact between animals, thus limiting the spread of sexually transmitted diseases. Successful semen cryopreservation enhances these advantages of AI over natural breeding. Long-term storage facilitates semen transport over distances, permits the quarantine of semen, and enables extended use of superior germplasm, even after the sire’s death. For agriculturally important animals, semen cryopreservation is an established industry worldwide, particularly for dairy cattle. Genome resource banking to preserve the biodiversity of endangered species or valuable transgenic lines also would benefit from sperm cryopreservation. For many mammals; however, effective semen cryopreservation is not a reality because a large number of sperm are apparently infertile following freezing and thawing. Compared to fresh, 8 times more cryopreserved bovine sperm were required to achieve equivalent fertilization rates in vivo (Shannon and Vishwanath, 1995). Although AI in pigs with fresh, cooled semen is increasingly popular, the fertility of cryopreserved semen remains about half that of fresh semen (Crabo, 1991). Following either transuterine or oviductal insemination of superovulated ewes, conception rates and percentage of fer-

The effect of reactive oxygen species on equine sperm motility, viability, acrosomal integrity, mitochondrial membrane potential, and membrane lipid peroxidation.

Abstract: The objective of this study was to examine the influence of reactive oxygen species (ROS), generated through the use of the xanthine (X)-xanthine oxidase (XO) system, on equine sperm motility, viability, acrosomal integrity, mitochondrial membrane potential, and membrane lipid peroxidation. Equine spermatozoa were separated from seminal plasma on a discontinuous Percoll gradient, and spermatozoa were incubated with 0.6 mM X and 0.05 U/mL XO for 30 minutes. Catalase (150 U/mL), superoxide dismutase (SOD, 150 U/mL), or glutathione (GSH, 1.5 mM) were evaluated for their ability to preserve sperm function in the presence of the induced oxidative stress. At the end of the 30-minute incubation, sperm motility was determined by computer-assisted semen analysis. Viability and acrosomal integrity were determined by Hoechst-Pisum sativum staining, and mitochondrial membrane potential was determined by staining with JC-1. Incubation with the X-XO system led to a significant (P < .01) increase in hydrogen peroxide production and an associated decrease (P < .01) in motility parameters. Total motility was significantly (P < .01) lower in the presence of X-XO compared with the case of the control (29%+/-9% vs 73%+/-1%, respectively). Catalase, but not SOD, prevented a decline in motility secondary to oxidative stress (71%+/-4% vs 30%+/-3%, respectively). The addition of glutathione had an intermediate effect in preserving sperm motility at the end of the 30-minute incubation (53%+/-3%). No influence of X-XO could be determined on viability, acrosomal integrity, or mitochondrial membrane potential. In order to promote lipid peroxidation, samples were incubated with ferrous sulfate (0.64 mM) and sodium ascorbate (20 mM) for 2 hours after the X-XO incubation. No increase in membrane lipid peroxidation was detected. This study indicates that hydrogen peroxide is the major ROS responsible for damage to equine spermatozoa. The decrease in sperm motility associated with ROS occurs in the absence of any detectable decrease in viability, acrosomal integrity, or mitochondrial membrane potential or of any detectable increase in lipid peroxidation.

Semen quality and human fertility: a prospective study with healthy couples.

Abstract: Measures of semen quality are used as surrogate measures of male fertility in clinical andrology, reproductive toxicology, epidemiology, and risk assessment. However, only limited data are available to relate those measures to fertility. This prospective study with 210 reproductive-age couples was conducted to provide information on the value of semen quality measures for predicting human male fertility potential and for development of models to estimate the effects of changes in semen quality on fertility in a given population for risk assessment. Couples without known risk factors for infertility and who had discontinued contraception to have a child were accepted. The study followed each couple for up to 12 menstrual cycles while they attempted to conceive and evaluated semen quality measures from multiple ejaculates per man with known abstinence intervals. For each cycle, the day of ovulation was predicted, and the couple was advised to have intercourse multiple times on that day and on the days around it. Among the demographic variables assessed, parity, contraception status prior to entering the study, male education level, and male smoking were associated significantly with 12-cycle pregnancy rate. Several semen quality measures also were associated significantly with pregnancy rate, with percentage morphologically normal sperm by strict criteria and measures involving total number of sperm showing particularly strong associations. Localized regression-smoothing plots of semen quality data against proportion of couples pregnant suggested levels below which fertility declines for several semen quality measures. These results have applications in both clinical andrology and in assessment of risk to male fecundity from environmental or pharmaceutical exposures. In particular, they contribute information on behavior of fertility with varying semen quality and can allow development of models to predict effects on fertility in populations from decrements in semen quality.

Functional and Ultrastructural Features of DNA‐Fragmented Human Sperm

Abstract: The functional significance of deoxyribonucleic acid(DNA) fragmentation in ejaculated human sperm is unclear. In thisstudy the extent of DNA strand breakage in swim-up selectedsper-matozoa was evaluated by terminal deoxynucleotidyl transferase-mediated fluorescein-dUTP nick end labeling (TUNEL)-coupledflow cytometry and correlated with several functional and morpho-logical sperm parameters. The extent of DNA fragmentation(mean 11.07% 8.00%, range 0.79%–42.64%, n 140) was pos-itively related to abnormal morphology and associated with defectsof the sperm tail. A negative correlation was found between DNAbreakage and progressive motility. When a stepwise multiple linearregression model was used to analyze the relationship betweenDNA fragmentation and the aforementioned parameters, only mo-tility results were included in the model. The presence of sper-matozoa showing submicroscopic characteristics resemblingthoseof somatic apoptosis has been reported in human ejaculate. Toverify whether sperm DNA fragmentation was associated with thepresence of such apoptotic-like cells, we performed electron mi-croscopy and TUNEL-coupled flow cytometry in a limited numberof sperm samples (n 24). Although we did not observe any sig-nificant relationship between DNA breakage and the characteris-tics that are suggestive of apoptosis, an association was foundwithseveral ultrastructural features, indicating an impaired motility.Hence, we conclude that in ejaculated sperm, DNA fragmentationdoes not correspond to the apoptosis-like phenomenon and that itis associated with defects of motility.Key words: Apoptosis, DNA fragmentation.J Androl 2000;21:903–912

Comparison of zinc concentrations in blood and seminal plasma and the various sperm parameters between fertile and infertile men.

Abstract: The aim of the study was to examine the relationships between concentrations of zinc in blood and seminal plasma and sperm quality among infertile and fertile men. One hundred seven male (infertile group) partners of couples who were undergoing investigation for infertility with no known cause for the infertility and 103 men (fertile group) whose wives were pregnant at the time of the study were recruited. The subjects' blood and seminal plasma concentration of zinc were determined by atomic absorption spectroscopy. Except for semen volume, all the other semen parameters for the infertile men were significantly lower than those for the fertile group. The geometric means of the seminal plasma zinc concentration were significantly lower in the infertile group compared with those in the fertile group; 183.6 mg/L (range, 63-499) versus 274.6 mg/L (range, 55-420). There were no significant differences in the geometric means of the blood zinc concentration between the 2 groups. Seminal plasma zinc concentration was significantly correlated with sperm density (r = 0.341, P < .0001), motility (r = 0.253, P < .0001), and viability (r = 0.286, P < .0001). On the basis of the findings of this study and those of other reports, zinc may contribute to fertility through its positive effect on spermatogenesis.

Morphologic Changes in Efferent Ductules and Epididymis in Estrogen Receptor‐α Knockout Mice

Abstract: Estrogen has been shown to have an important role in fluid reabsorption in efferent ductules of the testis. Our previous study of the estrogen receptor-alpha knockout mouse (ERKO) showed that the efferent ductules and rete testis were primary targets of estrogen receptor function. In the present study, a more comprehensive evaluation of the ERKO male reproductive tract was performed to determine the severity of effects in efferent ductules as well as the epididymis. The following observations were found in ERKO males: 1) blind-ending efferent ductules were more prevalent in ERKO than in wild type (WT) tissues; 2) glycogen-containing cells were observed at the rete testis-efferent ductule junction; 3) the tubular diameters of the efferent ductules and initial segment epididymides were dilated; 4) efferent ductules were dilated between 130 to 300% over wild type ductules; 5) efferent ductule epithelial height was reduced nearly 50%; 6) microvilli of nonciliated cells of efferent ductules were 64% shorter in length; 7) cilia were reduced in number; 8) initial segment epithelium was displaced into regions adjacent to the rete testis and in short segments of the common region of efferent ductule; 9) apical, narrow, and clear cells of the epididymis also were abnormal in some regions; 10) in the corpus and cauda regions, sperm granulomas were noted in one third of the ERKO males. In conclusion, the entire reproductive tract is affected in ERKO males. The cells showing the greatest effects were estrogen receptor-positive cells. It appears that in the ERKO mouse there are developmental anomalies that must be considered separately from adult dysfunctional changes in the male reproductive tract.

Characteristics of human sperm chromatin structure following an episode of influenza and high fever: a case study.

Abstract: Semen samples from a fertile patient presenting with influenza and a 1-day fever of 39.9 degrees C were obtained and analyzed at 18-66 days postfever (dpf) for sperm nuclear proteins, DNA stainability, free thiols (-SH), and susceptibility to DNA denaturation in situ. At 18 dpf, 36% of sperm demonstrated denatured DNA as measured by the sperm chromatin structure assay (SCSA), and decreased to 23% by 39 dpf. Samples at 33 and 39 dpf contained 49% and 30%, respectively, of cells with increased DNA stainability (HIGRN). A unique sperm nuclear protein band migrating between histones and protamines on acid-urea gels appeared at 33 and 39 dpf and nearly disappeared by 52 dpf. Amino acid sequencing of the first 8 N-terminal residues identified this protein as the precursor to protamine 2. The protamine P1 and P2 ratio remained normal, whereas the histone to protamine ratio increased slightly at 33 to 39 dpf. Flow cytometric measurements of nuclear -SH groups revealed the greatest reduction in free nuclear thiols at 33 dpf, and returned to normal by 45 dpf. The time of appearance of the unprocessed protamine 2 precursor and the relative increase in histone suggest a fever-related disruption of the synthesis of mRNA that codes for a P2 processing enzyme or enzymes. Increased DNA staining is likely due to the increased histone/protamine ratio. This case study demonstrates that fever/influenza can have latent effects on sperm chromatin structure and may result in transient release of abnormal sperm.

Peyronie's disease: Etiology, medical, and surgical therapy

Abstract: Peyronie's disease remains an enigma With the recent introduction of an animal model for Peyronie's disease, the entry of a number of double-blind placebo-controlled clinical trials, and the application of new molecular diagnostic methods, the investigation of this wound-healing disorder of the penile tunica albuginea should illuminate many of the unknowns Investigators need to be open to innovations in other fields of medicine involving idiopathic fibrosing conditions in other organ systems, eg, Dupuytren's contracture, keloids, hypertrophic scarring, etc Applications from these other disciplines will undoubtedly widen our scope about Peyronie's disease While a minority of patients respond with observation alone, most authorities recommend at least a trial of medical therapy with a safe, inexpensive, and well-tolerated agent, as early-stage disease is reputedly more likely to respond better than patients with established, longstanding Peyronie's plaques The reintroduction of intralesional therapies (verapamil and interferon alpha-2b) provides the clinician with an alternative minimally invasive intervention that has promising possibilities In severe fibrotic or calcified plaques or with major structural abnormalities, the judicious use of surgery with or without grafting materials and a penile prosthesis can restore many men back to their previous level of high esteem and provide both partners an excellent quality of life

Sperm preparation methods.

Abstract: DAVID MORTIMERFrom the Genesis Fertility Centre, Vancouver, BC,Canada.The spermatozoa of all placental (eutherian) mammals,including humans, are in a protective, nonlabile state atejaculation and are incapable of fertilization even if theyare placed in direct contact with an oocyte. Consequently,they must undergo a subsequent period of final maturationduring which they acquire the capacity to interact withthe oocyte–cumulus complex and achieve fertilization.This process, which was discovered independently byAustin and Chang in 1951, was termed capacitation, andspermatozoa in the ejaculate are prevented from under-going capacitation by one or more decapacitation factorsthat are present in the seminal plasma (Yanagimachi,1994). Capacitation of eutherian spermatozoa is essentialfor fertilization not only in vivo but also in vitro, andunderlies the manipulation of spermatozoa for clinical invitro fertilization (IVF).Not only does seminal plasma contain one or more de-capacitation factors that prevent spontaneous capacitationof spermatozoa upon ejaculation, but it also contains oneor more factors to which prolonged exposure has adverseeffects on sperm function, including the ability to pene-trate cervical mucus (Kremer, 1968), undergo the acro-some reaction in vitro, and the fertilization process in gen-eral (Rogers et al, 1983; Mortimer and Mortimer, 1992;Mortimer et al, 1998). Consequently, in order for euthe-rian spermatozoa to have the capacity to fertilize an oo-cyte, they must be separated from the seminal plasma,and hence, the separation of human spermatozoa fromseminal plasma is an essential prerequisite for them to beable to achieve capacitation and express their intrinsicfertilizing ability. In assisted reproductive technology(ART) laboratories, this need is manifested in the processcommonly referred to as ‘‘sperm washing,’’ in whichspermatozoa are somehow removed from the seminalplasma and resuspended in culture medium.Prolonged exposure ( 30 minutes) to seminal plasmaafter ejaculation can permanently diminish the fertilizingcapacity of human spermatozoa in vitro (Rogers et al,1983), and contamination of prepared sperm populationswith only traces of seminal plasma can diminish, or even

Cryopreservation alters the levels of the bull sperm surface protein P25b.

Abstract: Fertility of frozen-thawed bull sperm is reduced by cryopreservation. Freezing-thawing procedures can result in as much as a sevenfold fertility decrease. Sperm mortality and loss of motility do not fully explain the reduced fertility of cryopreserved semen; they may be partially explained by the loss of sperm surface proteins, which are necessary for fertilization. We have previously identified P25b, a sperm surface protein, which is associated with the fertility index of bulls used for artificial insemination. Using Western blotting techniques, we have evaluated P25b levels before and after cryopreservation of bull spermatozoa in extenders based on either egg yolk or milk. Long storage periods (28 days) in liquid nitrogen results in a threefold decrease of P25b levels associated with cryopreserved versus fresh spermatozoa. Over a short storage period (3-7 days), a stable P25b level was observed on spermatozoa cryopreserved in extender containing either egg yolk or milk. A decrease in P25b levels associated with spermatozoa was observed after 5 days of storage in egg yolk extender, whereas a significant decrease was observed after 14 days of sperm storage in milk extender (P < .05). Therefore, the loss of P25b may be responsible, at least in part, for the decrease in fertility following the freezing-thawing procedure of bull semen. Moreover, the cryopreservation extender used may have different effects on the loss of sperm surface proteins after even brief storage periods in liquid nitrogen. Considering that a sperm protein similar to P25b exists in humans (P34H), these results may have significant clinical applications in which frozen semen is used.

Human glyceraldehyde 3-phosphate dehydrogenase-2 gene is expressed specifically in spermatogenic cells.

Abstract: Although the process of glycolysis is highly conserved in eukaryotes, several glycolytic enzymes have unique structural or functional features in spermatogenic cells. We previously identified and characterized the mouse complementary DNA (cDNA) and a gene for 1 of these enzymes, glyceraldehyde 3-phosphate dehydrogenase-s (Gapds). This gene is expressed only in spermatids. The enzyme appears to have an essential role in energy production required for fertilization, and it is reported to be susceptible to inhibition by certain environmental chemicals. We have now cloned and sequenced the cDNA for the human homologue of glyceraldehyde 3-phosphate dehydrogenase (GAPD2) and determined the structure of the gene. The messenger RNA (mRNA) was detected in testis, but not in 15 other human tissues analyzed by Northern blot technique. The deduced GAPD2 protein contains 408 amino acids and is 68% identical with somatic cell GAPD. GAPD2 has a 72-amino acid segment at the amino terminal end that is not present in somatic cell GAPD. This segment is proline-rich but contains smaller stretches of polyproline and is 30 amino acids shorter than the comparable segment of mouse GAPDS. The structure of the human GAPD2 gene was determined by polymerase chain reaction (PCR) to identify exon-intron junctions in a genomic clone and in total genomic DNA. The locations of these junctions in the GAPD2 gene corresponded precisely to those of the 11 exon-intron junctions in the mouse Gapds gene. Immunohistochemical studies found that GAPD2 is located in the principal piece of the flagellum of human spermatozoa, as are GAPDS in mouse and rat spermatozoa. GAPD2 extracted from human spermatozoa and analyzed by Western blot technique migrated with an apparent molecular weight of approximately 56,000, although the calculated molecular weight is 44 501. The conserved nature of the mouse, rat, and human enzymes suggests that they serve similar roles in these and other mammalian species.

Long-term effects of triptolide on spermatogenesis, epididymal sperm function, and fertility in male rats.

Abstract: Prior studies had suggested that triptolide, a diterpene triepoxide isolated from a Chinese medicinal plant, might be an attractive candidate as a post-testicular male contraceptive agent. Despite the promise that triptolide would not affect testis function, nagging concerns remained that a delayed onset of testicular effect might exist. The objectives of this study were to assess the effects of relatively longer treatment duration of triptolide on fertility, spermatogenesis, and epididymal sperm pathophysiology; and to evaluate the reversibility of these effects after the cessation of treatment. Adult male Sprague-Dawley rats were fed daily with either 30% gum acacia as a vehicle control (n = 12) or 100 microg/kg body weight (BW) of triptolide for 82 days (n = 12) followed by a recovery period of up to 14 weeks (n = 6). At the end of the treatment period, all rats treated with triptolide were sterile. Cauda epididymal sperm content decreased by 84.8% and sperm motility was reduced to zero. In addition, virtually all cauda epididymal sperm in the triptolide-treated group exhibited severe structural abnormalities. The most striking changes observed were head-tail separation, premature chromatin decondensation of sperm nuclei, a complete absence of the plasma membrane of the entire middle and principle pieces, disorganization of the mitochondrial sheath, and aggregation of many sperm tails. Longer treatment duration of triptolide also affected spermatogenesis, with marked variability in the response of individual animals. The degree of damage ranged from apparently normal-looking seminiferous tubules to flattened seminiferous epithelium lined by a single layer of cells consisting of Sertoli cells and a few spermatogonia. Affected tubules exhibited intraepithelial vacuoles of varying sizes, multinucleated giant cells, germ cell exfoliation, and tubular atrophy. Recovery occurred as early as 6 weeks after cessation of treatment. By 14 weeks, 4 out of 6 triptolide-treated males were fertile and the females that were impregnated by 3 out of 4 triptolide-treated male rats produced apparently normal litters. These results suggest that triptolide has 2 phenotypic effects on mature and maturing germ cells. The first action appears earlier and manifests mainly in epididymal sperm. The second action presumably is directly on germ cells in testis and causes a variable impairment of spermatogenesis that may not be completely reversible. It is unclear if the earlier effect is a delayed manifestation of subtle testicular injury or post-testicular action.

Spontaneous hyperplasia of the ventral lobe of the prostate in aging genetically hypertensive rats.

Abstract: Recent studies have shown that the prostatic autonomic innervation takes part in its homeostasis and growth. Other works showed that spontaneously hypertensive rats (SHR) show excessive sympathetic activity, accompanied by lower urinary tract symptoms, increased growth capacity of prostatic stromal cells, and increased levels of androgens and their receptors. Furthermore, young SHR were reported to present incipient stages of benign prostatic hyperplasia (BPH). The aim of the present study was to examine whether this strain indeed develops spontaneous BPH with age, and can thus serve as a genuine natural model for this disorder. For this purpose, ventral lobes of prostates of one-year-old, male SHR and their normotensive counterparts, Wistar Kyoto (WKY) rats, were examined histopathologically, and the degree of hyperplasia was evaluated according to a score-chart protocol (histoscore). SHR exhibited severe adenomatous spontaneous BPH, characterized by piling-up of epithelial cells, with papillary formations, accompanied by a mild increase in the amount of fibrocytes and smooth muscle cells in the stroma. This was reflected by histoscore values of 38 +/-2. Thickening of prostatic arterioles also was noted, as well as mild chronic inflammatory exudate. WKY rats did not show any of these features of BPH despite their age (histoscore 17 +/- 3, significantly different from that of SHR). We conclude that SHR can serve as a rodent model for the spontaneous development of BPH with age, most probably due to the excessive neuroendocrine activity characteristic of this rat strain.

The Cyclic GMP-Specific Phosphodiesterase Inhibitor, Sildenafil, Stimulates Human Sperm Motility and Capacitation but Not Acrosome Reaction

Abstract: Capacitation is the series of transformations that spermatozoa undergo in the female genital tract in order to bind to the zona pellucida, initiate the acrosome reaction, and fertilize an egg. Cyclic adenosine monophosphate (cAMP) plays an important role in this process and its levels are regulated by 2 key enzymes, adenylyl cyclase and cyclic nucleotide phosphodiesterase (PDE), the latter being involved in cAMP degradation. Evidence was provided for the involvement of PDE in sperm motility and capacitation. Of the 10 gene families of PDE that exist in mammalian tissues, the calcium-calmodulin-dependent (type 1) and the cAMP-specific (type 4) have been found in human spermatozoa. Using sildenafil, we investigated a highly potent cyclic guanosine monophosphate (cGMP)-specific PDE (type 5) inhibitor and whether this PDE is present in human spermatozoa and is involved in sperm functions. Sildenafil inhibited PDE activity of Percoll-washed spermatozoa with an IC 50 of 97 ± 3 and 33 ± 3 μM when cAMP and cGMP, respectively, were used as substrates. Because the IC 50 of sildenafil obtained for PDE type 5 is much lower (2 to 6 nM) than that obtained with sperm PDE, the data suggest that PDE type 5 represents only a small fraction of the whole PDE activity of spermatozoa. Sildenafil causes dose-dependent increases in sperm cAMP levels and capacitation, which are associated with an increase in the levels of tyrosine phosphorylation of 2 fibrous sheath proteins (p105/81). Sperm velocity, amplitude of lateral head displacement, and hyperactivation were increased at 30-180 minutes. Sildenafil did not trigger the acrosome reaction in capacitated spermatozoa. These results suggest that under our experimental conditions, sildenafil triggers human sperm motility and capacitation, probably via its inhibitory action on PDE activity other than type 5 with a resultant rise in cAMP levels.

Sperm Calcium Levels and Chlortetracycline Fluorescence Patterns are Related to the In Vivo Fertility of Cryopreserved Bovine Semen

Abstract: Cryopreserved bovine semen is less fertile than fresh semen for reasons that have not been fully elucidated. Cryopreservation is known to disrupt the sperm plasma membrane and it induces premature capacitation of a sperm subpopulation, which may be a result of the increased internal calcium levels after thawing. To test the hypothesis that sperm intracellular calcium level is correlated with in vivo fertility, we used the fluorescent calcium indicator, indo-1, and flow cytometry to assess intracellular calcium levels in frozen-thawed sperm from bulls of varying degrees of fertility. We also tested a second hypothesis that the physiological status of sperm, as assessed by the chlortetracycline (CTC) fluorescent assay, is correlated with fertility. As detected by indo-1 fluorescence, the intracellular calcium level is negatively correlated with bull fertility immediately after thawing (P = .0362; n = 3 ejaculates from each of 10 animals). Moreover, there was a significant difference between the 3 most and least fertile bulls over 4 hours of incubation (P < .05; n = 3 ejaculates per bull). Finally, there was a positive correlation between sperm displaying the CTC acrosome reaction pattern and fertility (P = .0014; n = 3 ejaculates from each of 10 bulls).

Phenotypes of sperm pathology: genetic and acquired forms in infertile men.

Abstract: Asthenozoospermia and teratozoospermia are frequently responsible for infertility in men, yet they are poorly understood conditions that are often unrelated to any known andrological disorder. The cause of male infertility remains unclear in many individuals and numerous patients are believed to suffer from idiopathic infertility; however, recent developments have demonstrated that genetic abnormalities play a major role. The classic description by Scandinavian groups in the mid 1970s that absolute asthenozoospermia could be caused by genetic-related dynein (a structural protein with ATPase activity) deficiency in spermatozoa (Afzelius et al, 1975; Pedersen and Rebbe, 1975) opened up a new field that has been subsequently enriched by the recognition that other alterations such as congenital absence of the vas and classical forms of cystic fibrosis and, more recently, microdeletions in the long arm of the Y chromosome, can be responsible for male infertility. In the present review I will develop the concept that various forms of asthenozoospermia or teratozoospermia are also of genetic origin. Spermatozoa are terminally differentiated cells with organelles that are specialized in particular functions. They have a pathology of their own, which is not identified by routine semen analysis or functional tests because the deficiencies demonstrated by these methods do not reveal the underlying pathology but, rather, are secondary manifestations. The powerful resolution of electron microscopy overcomes the limitations of light microscopy and allows an excellent observation of sperm pathology because the internal Supported by grants from CONICET (PICT 0090) and ANPCyT (PICT

Posttesticular antifertility action of triptolide in the male rat: evidence for severe impairment of cauda epididymal sperm ultrastructure.

Abstract: A variety of active diterpene epoxides, including the triptolide (isolated from Tripterygium wilfordii) have been reported to cause infertility in male rats. Previously, we showed that oral admin- istration of triptolide at a dosage of 100 g/kg per body weight for 70 days completely inhibited fertility in male rats, with little or no demonstrable detrimental effect on spermatogenesis and Leydig cell function as determined by testicular light microscopic appearance and serum and intratesticular testosterone levels. Despite the ap- parent absence of effects on the testes, cauda epididymal sperm were abnormal, with complete cessation of sperm motility and some reduction in sperm numbers. This study was undertaken to provide additional insight into the subcellular sites and possible mechanisms of action of this compound using ultrastructural analysis of the testes and epididymidis. The most striking effect of triptolide treatment was observed in sperm in the epididymis. In rats rendered infertile with 100 g/kg per body weight of triptolide daily for 70 days, virtually all cauda epididymal sperm exhibited complete absence of plasma membrane over the entire middle and principal piece, premature de- condensation of the nuclei, and disorganization of the mitochondrial sheath with many vacuolated mitochondria. No ultrastructural differ- ences in the epididymal epithelium were observed between control and triptolide-treated rats. The testes appeared to be mildly affected after triptolide treatment but exhibited only subtle ultrastructural de- fects in the germ cells. The findings of severe impairment of cauda epididymal sperm ultrastructure, along with minimal discernible ab- normalities in the fine structural cytology of the testes, further sug- gest that the site of action of this compound is posttesticular and may be confined to the cauda epididymal sperm. However, we can- not rule out an effect of triptolide that occurs during germ cell mat- uration but is delayed in its manifestation or triggered at the rete testis and epididymal level.

Oxidative stress in prostatic fluid of patients with chronic pelvic pain syndrome : Correlation with Gram positive bacterial growth and treatment response

Abstract: The etiology of chronic pelvic pain syndrome (CPPS)/chronic prostatitis category III remains unknown. Whereas a subset of men respond to antimicrobial therapy, gram positive bacteria isolated from expressed prostatic secretions (EPS) are often considered to be commensal rather than pathogenic. We wished to study oxidative stress as a marker of tissue injury and response in EPS of men with CPPS to determine whether infection with gram positive bacteria is associated with increased oxidative stress. A total of 300 EPS specimens from 100 men with CPPS were collected for microscopy, culture, and biochemical and molecular assays. Oxidant injury was measured by 8-isoprostane F2alpha (IsoP) levels and total antioxidant capacity as Trolox equivalents. Total RNA from EPS was used for gene expression of heme oxygenase-1 (HO-1) and granzyme B. The only bacteria found in EPS were gram positive. For our analysis, these men were classified as having chronic bacterial prostatitis (category II). IsoP levels (pg/mL) were highest in men with category II prostatitis (7315 +/- 1428) followed by nonbacterial prostatitis (category IIIa, 2043 +/- 561), prostatodynia (category IIIb, 319 +/- 81), and asymptomatic controls (298 +/- 99). IsoP levels decreased significantly after successful treatment with antibiotics or an antioxidant supplement (Prosta-Q). Antioxidant capacity was detected in 11 out of 18, 4 out of 16, and 1 out of 16 men tested with category II, IIIa, and IIIb prostatitis, respectively. No correlation was observed between IsoP levels and the number of white blood cells in EPS. HO-1 and granzyme B expression was highest in men with category II prostatitis than in men with either category III prostatitis or asymptomatic controls. On the basis of elevated oxidative stress, clinical response to antibiotics, and post-treatment reduction in oxidative stress, we conclude that gram positive bacteria in some men with CPPS may be pathogens. It is speculated that oxidative stress may be a key pathway in some men with CPPS that can be targeted with antioxidant therapy.

Correlation of CASA velocity and linearity parameters with sperm mobility phenotype in turkeys.

Abstract: Since all domestic turkeys are produced through artificial insemination, a measurable sperm characteristic that would be predictive of fertility would allow for the culling of poor males, resulting in improved reproductive efficiency. The sperm mobility test (SMT), which quantifies sperm penetration into an Accudenz solution, has been shown to correlate highly with fertilization potential of individual turkeys. Since this sire-selection test is based on the differences in sperm mobility between whole ejaculates from individual males, the objective of this study was to determine whether specific sperm velocity parameters would correlate with the SMT and to determine whether these characteristics could account for phenotypic differences in sperm mobility observed between males. The SMT was used to rank males within a flock (n = 110) in triplicate and to classify them into high, average, and low sperm mobility phenotypes on the basis of the sperm mobility index. Several sperm velocity parameters were evaluated for each male by a computer-aided sperm analysis (CASA) system, the Hobson Sperm Tracker. The types of measurements taken of 200 sperm tracks/ejaculate included the following: curvilinear velocity (VCL), average path velocity (VAP), straight-line velocity (VSL), linearity (LIN), beat-cross frequency (BCF), and mean angular displacement (MAD). Significant positive correlations were found between VSL, LIN, BCF, and sperm mobility, and a significant negative correlation was seen between MAD and sperm mobility. Subpopulations of sperm that had penetrated the Accudenz solution were isolated from each mobility phenotype and were analyzed by CASA, and significant correlations were again observed between VSL, LIN, BCF, and sperm mobility. We conclude that sperm velocity and linearity contribute to overall sperm mobility phenotype and are important characteristics of turkey sperm function. Key words: Motility, computer, spermatozoa.

Acidification of intracellular pH in bovine spermatozoa suppresses motility and extends viable life.

Abstract: Intracellular pH (pHi) was determined in ejaculated bovine spermatozoa using a ratiometric absorbance technique under various incubation conditions that drastically altered sperm motility. The pHi was directly correlated with sperm motility. In a medium of Sodium, Potassium, and Magnesium [NKM] that supported active sperm motility, pHi was 6.9. In medium containing weak acids (NKM equilibrated with 100% CO2 or containing 80 mM 5,5-dimethyl-2,4-oxazolidinedione; DMO), pHi was depressed at least 0.5 pH unit and sperm motility was suppressed. After complete immobilization of sperm was established, removal of the weak acids indicated that suppression of motility was fully reversible for up to 48 hours in CO2 and up to 24 hours in DMO. This study shows that expression and conservation of sperm motility are inversely related, and that depression of pHi by weak acids can reversibly inhibit sperm motility. These findings may help to explain the mechanisms by which sperm are immobilized within the male reproductive tract, and could be applicable to the design of improved ambient temperature semen extenders.

Glycerol disrupts tight junction-associated actin microfilaments, occludin, and microtubules in Sertoli cells.

Abstract: Intratesticular injections of glycerol have been shown to result in a marked and prolonged reduction of spermatogenesis, accompanied by increased permeability of the blood-testis barrier. Because the permeability of the blood-testis barrier is regulated by Sertoli cell tight junctions, and tight junction organization is regulated by the cytoskeleton, we undertook to examine the effects of glycerol treatment on cytoskeletal actin microfilaments and microtubules, and on the tight junction protein, occludin, in Sertoli cells. Adult rats received a single intratesticular injection of either saline (controls) or a 10% glycerol solution. At 24 hours and 7, 15, and 21 days after injection, testes were collected and prepared for routine histology, cryosectioning, or whole seminiferous tubule immunohistochemical staining; and the preparations were viewed by light and confocal microscopy. In saline-injected testes, Sertoli cells had a cytoskeletal and junctional organization that resembled that of normal testes. F-actin microfilaments, located in the basal region, were arranged in regular bundles or chords that circumscribed the perimeter of each Sertoli cell at the level of the tight junction. Occludin colocalized with tight junction-associated actin filament distribution and microtubules formed a geometric array associated with spermatogenic cells. In contrast, in glycerol-treated Sertoli cells, microfilament and microtubule organization and occludin distribution were partially or completely disrupted. From these results we conclude that glycerol treatment either directly or indirectly disrupts tight junction-associated F-actin and occludin and tubulin organization in rat Sertoli cells. Perturbation of the tight junction-associated proteins could explain the increase in permeability of the blood-testis barrier observed after glycerol treatment. Impaired spermatogenesis following glycerol treatment is likely a consequence of a leaky blood testis barrier and disrupted Sertoli cell cytoskeleton. Glycerol injections may serve as a useful tool in studying the relationship between cytoskeletal organization and the stabilization of Sertoli-Sertoli cell junctions.

Screening for AZF Deletion in a Large Series of Severely Impaired Spermatogenesis Patients

Abstract: Recent investigations have pointed to a high prevalence of Y chromosome submicroscopic deletions in men with severely im- paired spermatogenesis. We report on the incidence in 128 infertile men, in whom karyotype, sperm count, and hormonal parameters were evaluated. Patients with abnormal karyotype (other than an ab- normal Y chromosome) or sperm concentration of more than 2 million/ mL were excluded. Genomic DNA was extracted from the peripheral leukocytes of 57 men with azoospermia and 71 with severe oligo- spermia. Molecular analysis was performed by 3 multiplex polymerase chain reactions using a set of 9 sequence tagged sites (STSs) from 3 different regions of the Y chromosome: AZFa, AZFb, and AZFc. In 7% of the studied patients Yq microdeletions were detected, with a high prevalence in men with azoospermia (14%). No deletions were detected in the AZFa region. Deletions were present in AZFb, AZFc, or both regions. The deletion observed in 1 patient that did not overlap with the DAZ region demonstrates that genes other than DAZ may also be involved in the pathogenesis of some subsets of male infer- tility. Furthermore, common Yq deletions present different testicular pictures, suggesting that some unknown factors may be disturbing spermatogenesis. Because men with severe infertility suffer a high risk of Y chromosome deletion, screening for these men is recommended prior to treatment with assisted reproduction.

Quantification of the nonenzymatic fast and slow TRAP in a postaddition assay in human seminal plasma and the antioxidant contributions of various seminal compounds.

Abstract: Total radical-trapping antioxidant potential (TRAP) measurements of human seminal plasma (N = 25) were performed by using a post-addition assay based on trapping 2,2' Azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) radicals. This method enables the antioxidant capacity of human seminal plasma and its constituents to be quantified. The standard procedure consisted of determination of the Trolox equivalent antioxidant capacity (TEAC) after incubating the test sample in the ABTS radical solution for 10 seconds (fast TRAP) and 300 s (total TRAP). Interestingly, seminal plasma showed a fast TRAP and a high slow TRAP (Total TRAP - Fast TRAP). The final total TRAP of seminal plasma is about 10 times higher than that of blood plasma. Various components of seminal plasma contribute to its fast TRAP; 37% can be attributed to vitamin C, uric acid, and tyrosine; proteins and polyphenolic compounds contribute a further 57%. In contrast, the slow TRAP was attributed to vitamin C (1%), uric acid (2%), and tyrosine (15%) and to proteins and polyphenolic compounds (33%). It was not possible to account for the remaining 49%. Neither known putative antioxidants, such as spermine, pyruvate, and taurine, nor other seminal compounds, such as carnitine, sialic acid, fructose, spermidine, glycerophosphorylcholine, and hyaluronic acid, contributed to any significant radical-trapping activity at a standard concentration of 1 mM. Of the amino acids, only tyrosine possessed a slow TRAP, and it is present at a high concentration in seminal plasma. Glutathione and hypotaurine show high fast and slow TRAPs, respectively. However, because of their low concentration in seminal plasma, their contribution to the TRAP is negligible. In conclusion, seminal plasma possesses a high antioxidant buffer capacity that protects spermatozoa from oxidative stress. Moreover, these findings suggest that the fast and slow TRAPs may have an important role as infertility markers and treatment targets in future antioxidant therapies.

Immunolocalization of CA II and H+ V-ATPase in epithelial cells of the mouse and rat epididymis.

Abstract: Acidification of the epididymal lumen has been suggested to play an important role in sperm functions; however, the cell types, pumps, and mechanisms involved have not been fully addressed. In this study, carbonic anhydrase II (CA II) and a 67-kd subunit of Neurospora crassa vacuolar proton adenosinetriphosphatase (H+ V-ATPase) pump were immunolocalized using light microscopy and electron microscopy (EM) in the epididymis of rats and mice. In both animals, narrow cells, identified in the initial segment and intermediate zone of the epididymis, contained numerous small vesicles in their apical region, often cup-shaped in appearance. In the mouse but not rat, these cells also possessed numerous cisternae of smooth endoplasmic reticulum, suggesting steroid synthesis; and cytoplasmic blebs of their apical cell surface, which appeared to detach, suggesting apocrine secretion. Anti-CA II antibody was immunocytochemically localized in the light microscope within narrow cells but not over any other cell types of the entire epididymis. Anti-H+ V-ATPase antibody was also localized in narrow cells of the initial segment and intermediate zone; as well as clear cells of the caput, corpus, and cauda regions. Using EM, gold particles for anti-CA II and H+ V-ATPase antibodies were noted in the apical region of narrow cells in relation to the numerous, small, cup-shaped vesicles. Although CA II was mainly located in the cytosol near these vesicles, H+ V-ATPase appeared on their delimiting membrane and on the apical plasma membrane of these cells. A similar distribution was noted for H+ V-ATPase in clear cells. The nature of the small vesicles of the apical region of narrow cells was examined with electron-dense fluid phase tracers that were introduced into the epididymal lumen. The tracers appeared within these vesicles and a few endosomes 1 hour after injection, suggesting that they contact the apical plasma membrane. Since these vesicles are also related to CA II and H+ V-ATPase, the data suggests that, as the site of proton production, the vesicles recycle to and from the apical cell surface, and in this way, deliver protons to the epididymal lumen for acidification. Clear cells and their expression of H+ V-ATPase may also serve in this function. In summary, both narrow and clear cells appear to be involved in luminal acidification, an activity that may be essential for sperm as they traverse and are stored in the epididymis.

Transdermal electromotive multi-drug administration for Peyronie's disease: preliminary results.

Abstract: The purpose of this study was to clarify the actual therapeutic potential of a new transdermal drug delivery system (electromotive drug administration; EMDA) for selected patients with Peyronie's disease. Forty patients with Peyronie's disease were treated by electromotive administration of the 3-drug association orgotein-dexamethasone-lidocaine in a double-blind, placebo-controlled, partial crossover study (study 1). Another 25 patients were treated by EMDA with a combination of verapamil-dexamethasone in an uncontrolled study (study 2). Treatment sessions lasted 20 minutes each and took place 3 times a week for 3 weeks with a current of 3 mA. Patients were assessed before treatment and at 1-and 3-month follow-up examinations. Assessments were based on sexual history, physical examination, and dynamic color Doppler ultrasonographic results. Adverse effects of EMDA were not reported. In study 1, the clinical results observed after treatment proved to be significantly better than those of the placebo. Penile pain disappeared in all patients in both studies. Penile lesion (nodule or plaque) either disappeared or significantly improved in 79% and 90% of patients treated by the 3- and 2-drug association, respectively. The improvement of penile deformity also was notable although it did not match the effect observed on penile nodules or plaque (62% and 88%, in studies 1 and 2, respectively). In both studies, more than 80% of patients reported a definite amelioration of penile rigidity, which paralleled the improvement of penile dynamic color Doppler e ultrasonographic parameters. Overall, the combination of verapamil-dexamethasone achieved better clinical results than the 3-drug combination. Electromotive drug administration is a novel technique capable of safely achieving satisfactory results in selected patients with Peyronie's disease not only in terms of improvement of patient's symptoms but also due to the reduced need for penile surgery.